We propose to undertake a molecular investigation of mutagenesis by DNA-modifying carcinogens. We will initiate our work with the potent mutagenic carcinogen aflatoxin B1 (AFB1) in a prokaryotic model system (phi chi 174-Escherichia coli). We will react AFB1, activated by enzymatic or chemical procedures, with phi chi DNA to produce two types of DNA-modifications: (a) random modifications of phi chi single-stranded and double-stranded DNA and (b) specific, characterized modifications in a pre-selected part of phi chi double-stranded DNA. We will use such modified DNA to transfect a variety of host strains with selected repair backgrounds, and isolate all possible types of mutations caused by AFB1 modifications. We will use the strategy of isogenic rescue recently developed by us to isolate non-conditional lethal mutants of phi chi. This simple, versatile strategy involves plasmid-mediated complementation of defective viral genes. We will characterize representative mutants to the nucleotide sequence level. Examples of the types of questions we hope to answer are the following: (1) Is the guanine N7 adduct, the principal product of AFB1 modification, mutagenic? (2) What types of mutations (point, frame-shift, deletion, etc.) are produced by AFB1 and in what proportion? (3) What are the mechanisms of AFB1-induced mutagenesis? Are nucleotide substitutions or insertions random or is there a pattern? Are frameshifts caused by deletions or insertions and if so how many nucleotides are involved? (4) What specific types of mutations are produced when AFB1 modifications are repaired by (a) excision pathway (b) error-prone, induced (SOS) repair pathway? (5) What are the enhancing and mitigating factors in AFB1-induced mutagenesis (e.g. nucleotide sequence, repair mechanisms)?